In the past, the resolution of key impurities such as Pneumocandins B5 and E0 from Pneumocandin B0 in the silica gel HPLC purification was poor. Some analogs were only partially resolved from the main product peak under preparative conditions. To achieve the desired product purity, the limited resolution required that the purification step be run at low column loading, which limited productivity.
Lipopeptides, such as Pneumocandin B0, are often the product of a fermentation process. During such a fermentation process, many very closely related analogues are produced along with the desired product. Normal phase chromatography systems are frequently used to purify the crude fermentation product. A normal phase chromatography system usually consists of a stationary phase and a mobile phase. For purification of a peptide or lipopeptide, the stationary phase can be silica gel or alumina, and the mobile phase can be a single solvent or a mixture of solvents, which includes organic solvents and water.
Silica gel chromatography and other types of normal phase chromatography are useful for separating these analogues. However, in practice, the resolution of certain close analogues from the desired product is often poor and not satisfactory, because the resolution is not great and often there is overlap. To achieve the desired purity of the main product at a reasonable yield requires restricting the amount of material (often referred to as feed or column load) loaded onto the column per run, which limits the productivity of the operation.
The purification of Pneumocandin B0 falls into this category. The chromatography utilizes a mobile phase consisting of a mixture of solvents, specifically ethyl acetate (EtOAc), methanol (MeOH) and water, on a silica gel column. Pneumocandin B0, with a molecular weight of 1065 Daltons, is a natural product and serves as an intermediate in the production of Caspofungin acetate (Cancidas®). Pneumocandin B0 is produced as a secondary metabolite by fermentation of the fungus Glarea lozoyensis. See U.S. Pat. Nos. 5,194,377 and 5,202,309. The structures of Pneumocandin B0 and two of the key analog impurities, all comprised of a cyclic hexapeptide coupled with dimethylmyristate side chain, are shown in Formula I and Table 1.

TABLE 1Pneumocandin B0 and two of its analogsCompoundR1R2R3R4R5R6Pneumocandin B0OHOHMeOHHOHPneumocandin B5OHHMeOHHOHPneumocandin E0OHOHMeOHHH
Silica gel chromatography exploits the subtle variations in binding affinity of the hydroxy-rich cyclic hexapeptide core of the desired product and the analog impurities, including Pneumocandins B5 and E0, to effect a separation. In the silica gel HPLC purification, Pneumocandins B5 and E0, two of the key analog impurities co-produced in the fermentation of Pneumocandin B0, elute very close to Pneumocandin B0. Therefore, to meet the target impurity levels in the purified material for these and similar analog compounds, the quantity of crude Pneumocandin B0 that can be loaded onto the column is limited. As a result, significant efforts have been made to improve the resolution of key impurities. For instance, the ternary ethyl acetate-methanol-water mobile phase has been balanced to optimize resolution between Pneumocandin B0 and key analog impurities. D. J. Roush, F. D. Antia, K. E. Göklen J. Chromatography A, 827 (1998) 373-389.